PROJECT SUMMARY Cancers, though heterogeneous in pathology, are a product of unchecked cell growth caused by perturbation to essential cellular processes, notably energy metabolism, DNA replication, or the cell cycle. Misregulation of these pathways can cause severe phenotypes that, together, can lead to oncogenesis: increased DNA content, multinucleation, supernumerary centrosomes, and increased dependence upon glycolysis as the cellular source of ATP (resulting in changes to mitochondrial morphology, gene expression, and functions). Even though these cancer hallmarks have been intensively studied, the mechanism by which lesions to these pathways promote oncogenesis remains unclear in most cases. My lab studies ARF family GTPases and the mechanisms that they employ to regulate a wide variety of essential cellular activities that, when perturbed, can lead to severe pathologies. I used CRISPR technologies to generate cell lines lacking expression of ELMOD2, a regulator of several members of the ARF family. I discovered that ELMOD2-/- lines have fragmented mitochondria, and they also have increases in mitotic indices, supernumerary centrosomes, increased DNA content, multinucleation, and multiciliation, consistent with a defect in the cell cycle. Thus, I hypothesize that ELMOD2 both acts from inside mitochondria to mediate effects of the ARL2 GTPase on mitochondrial fusion as well as regulates cytokinesis from a distinct cellular compartment. To tease apart the underlying mechanisms that drive these functions, I will use mammalian cell culture to visualize and assess mitochondrial activity and cell cycle. In Aim 1, I will determine if ELMOD2 acts downstream of ARL2 to regulate mitochondrial fusion, independent of its roles in cell cycle. I will perform a series of rescue experiments to characterize ELMOD2?s role in this pathway and its relationship to other players in mitochondrial fusion. In Aim 2, I will define ELMOD2?s functions in cell cycle. I will begin by teasing apart the stage of cell cycle that is perturbed by loss of ELMOD2. I will determine which ARF family GTPase(s) ELMOD2 works with to alter mitochondrial dynamics and cell cycle. Overall, this project will contribute to our fundamental understanding of mitochondrial fusion and cell cycle, two critical pathways whose regulation is incompletely understood. This will broaden our understanding of regulatory GTPase biology and how GAPs may facilitate multi-pathway communication. Such findings will pave the way for the future design of novel therapeutics that will precisely target the source of cancer and other pathologies.